Spatial scale influences the distribution of viral diversity in the eukaryotic virome of the mosquito Culex pipiens.

Our knowledge of the diversity of eukaryotic viruses has recently undergone a massive expansion. This diversity could influence host physiology through yet unknown phenomena of potential interest to the fields of health and food production. However, the assembly processes of this diversity remain elusive in the eukaryotic viromes of terrestrial animals. This situation hinders hypothesis-driven tests of virome influence on host physiology. Here, we compare taxonomic diversity between different spatial scales in the eukaryotic virome of the mosquito Culex pipiens. This mosquito is a vector of human pathogens worldwide. The experimental design involved sampling in five countries in Africa and Europe around the Mediterranean Sea and large mosquito numbers to ensure a thorough exploration of virus diversity. A group of viruses was found in all countries. This core group represented a relatively large and diverse fraction of the virome. However, certain core viruses were not shared by all host individuals in a given country, and their infection rates fluctuated between countries and years. Moreover, the distribution of coinfections in individual mosquitoes suggested random co-occurrence of those core viruses. Our results also suggested differences in viromes depending on geography, with viromes tending to cluster depending on the continent. Thus, our results unveil that the overlap in taxonomic diversity can decrease with spatial scale in the eukaryotic virome of C. pipiens. Furthermore, our results show that integrating contrasted spatial scales allows us to identify assembly patterns in the mosquito virome. Such patterns can guide future studies of virome influence on mosquito physiology.

Microbiota Variation Across Life Stages of European Field-Caught Anopheles atroparvus and During Laboratory Colonization: New Insights for Malaria Research.

The potential use of bacteria for developing novel vector control approaches has awakened new interests in the study of the microbiota associated with vector species. To set a baseline for future malaria research, a high-throughput sequencing of the bacterial 16S ribosomal gene V3-V4 region was used to profile the microbiota associated with late-instar larvae, newly emerged females, and wild-caught females of a sylvan Anopheles atroparvus population from a former malaria transmission area of Spain. Field-acquired microbiota was then assessed in non-blood-fed laboratory-reared females from the second, sixth, and 10th generations. Diversity analyses revealed that bacterial communities varied and clustered differently according to origin with sylvan larvae and newly emerged females distributing closer to laboratory-reared females than to their field counterparts. Inter-sample variation was mostly observed throughout the different developmental stages in the sylvan population. Larvae harbored the most diverse bacterial communities; wild-caught females, the poorest. In the transition from the sylvan environment to the first time point of laboratory breeding, a significant increase in diversity was observed, although this did decline under laboratory conditions. Despite diversity differences between wild-caught and laboratory-reared females, a substantial fraction of the bacterial communities was transferred through transstadial transmission and these persisted over 10 laboratory generations. Differentially abundant bacteria were mostly identified between breeding water and late-instar larvae, and in the transition from wild-caught to laboratory-reared females from the second generation. Our findings confirmed the key role of the breeding environment in shaping the microbiota of An. atroparvus. Gram-negative bacteria governed the microbiota of An. atroparvus with the prevalence of proteobacteria. Pantoea, Thorsellia, Serratia, Asaia, and Pseudomonas dominating the microbiota associated with wild-caught females, with the latter two governing the communities of laboratory-reared females. A core microbiota was identified with Pseudomonas and Serratia being the most abundant core genera shared by all sylvan and laboratory specimens. Overall, understanding the microbiota composition of An. atroparvus and how this varies throughout the mosquito life cycle and laboratory colonization paves the way when selecting potential bacterial candidates for use in microbiota-based intervention strategies against mosquito vectors, thereby improving our knowledge of laboratory-reared An. atroparvus mosquitoes for research purposes.

Late Pleistocene environmental changes lead to unstable demography and population divergence of Anopheles albimanus in the northern Neotropics.

We investigated the historical demography of Anopheles albimanus using mosquitoes from five countries and three different DNA regions, the mitochondrial cytochrome oxidase subunit I gene (COI), the single copy nuclear white gene and the ribosomal internal transcribed spacer two (ITS2). All the molecular markers supported the taxonomic status of a single species of An. albimanus. Furthermore, agreement between the COI and the white genes suggested a scenario of Pleistocene geographic fragmentation (i.e., population contraction) and subsequent range expansion across southern Central America.

Laboratory colonization and maintenance of Anopheles atroparvus from the Ebro Delta, Spain.

BACKGROUND: Historically, Anopheles atroparvus has been considered one of the most important malaria vectors in Europe. Since malaria was eradicated from the European continent, the interest in studying its vectors reduced significantly. Currently, to better assess the potential risk of malaria resurgence on the continent, there is a growing need to update the data on susceptibility of indigenous Anopheles populations to imported Plasmodium species. In order to do this, as a first step, an adequate laboratory colony of An. atroparvus is needed. METHODS: Anopheles atroparvus mosquitoes were captured in rice fields from the Ebro Delta (Spain). Field-caught specimens were maintained in the laboratory under simulated field-summer conditions. Adult females were artificially blood-fed on fresh whole rabbit blood for oviposition. First- to fourth-instar larvae were fed on pulverized fish and turtle food. Adults were maintained with a 10% sucrose solution ad libitum. RESULTS: An An. atroparvus population from the Ebro Delta was successfully established in the laboratory. During the colonization process, feeding and hatching rates increased, while a reduction in larval mortality rate was observed. CONCLUSIONS: The present study provides a detailed rearing and maintenance protocol for An. atroparvus and a publicly available reference mosquito strain within the INFRAVEC2 project for further research studies involving vector-parasite interactions.

Evidence of Zika virus horizontal and vertical transmission in Aedes albopictus from Spain but not infectious virus in saliva of the progeny.

Aedes albopictus mosquitoes have been experimentally demonstrated to be a competent vector for Zika virus (ZIKV) in different countries, but there are still some gaps related to the importance of Ae. albopictus in ZIKV transmission. Recent studies on Spanish Ae. albopictus populations showed controversial results for ZIKV transmission and no studies have been performed yet to detect infectious ZIKV in saliva of progeny of infected female mosquitoes. Herein, the horizontal transmission (HT) and vertical transmission (VT) of ZIKV in field-collected Ae. albopictus mosquitoes from Spain were evaluated for ZIKV strains (African I and Asian lineages) to better estimate the risk of ZIKV transmission by Ae. albopictus. The two field-collected Ae. albopictus populations assayed were infected by all tested ZIKV strains, however differences in terms of vector competence were detected depending on strain-population combination. Moreover, a higher susceptibility to the African I lineage strain than to the Asian lineage strain was observed in both mosquito populations. On the other hand, VT was demonstrated for both ZIKV lineages, detecting the virus in both males and females of the progeny of infected females, although importantly ZIKV dissemination and transmission were not detected in the infected females from the offspring. The results of the present study demonstrate that Spanish Ae. albopictus populations could sustain virus transmission in case of ZIKV introduction, but VT would play a poor role in the ZIKV epidemiology. Overall, our results provide helpful information to health authorities to establish efficient surveillance and vector control programmes for ZIKV.

Viromics on Honey-Baited FTA Cards as a New Tool for the Detection of Circulating Viruses in Mosquitoes.

Worldwide, emerging and re-emerging infectious diseases (EIDs) are a major burden on public and animal health. Arthropod vectors, with mosquitoes being the main contributors of global disease, transmit more than 70% of the recognized EIDs. To assess new alternatives for arthropod-borne viral diseases surveillance, and for the detection of new viruses, honey-baited Flinders Technology Associates (FTA) cards were used as sugar bait in mosquito traps during entomological surveys at the Llobregat River Delta (Catalonia, Spain). Next generation sequencing (NGS) metagenomics analysis was applied on honey-baited FTA cards, which had been exposed to field-captured mosquitoes to characterize their associated virome. Arthropod- and plant-infecting viruses governed the virome profile on FTA cards. Twelve near-complete viral genomes were successfully obtained, suggesting good quality preservation of viral RNAs. Mosquito pools linked to the FTA cards were screened for the detection of mosquito-associated viruses by specific RT-PCRs to confirm the presence of these viruses. The circulation of viruses related to Alphamesonivirus, Quaranjavirus and unclassified Bunyavirales was detected in mosquitoes, and phylogenetic analyses revealed their similarities to viruses previously reported in other continents. To the best our knowledge, our findings constitute the first distribution record of these viruses in European mosquitoes and the first hint of insect-specific viruses in mosquitoes' saliva in field conditions, demonstrating the feasibility of this approach to monitor the transmissible fraction of the mosquitoes' virome. In conclusion, this pilot viromics study on honey-baited FTA cards was shown to be a valid approach for the detection of viruses circulating in mosquitoes, thereby setting up an alternative tool for arbovirus surveillance and control programs.

Field-captured Aedes vexans (Meigen, 1830) is a competent vector for Rift Valley fever phlebovirus in Europe.

BACKGROUND: Aedes vexans (Meigen) is considered a nuisance species in central Europe and the Mediterranean region. It is an anthropophilic and mammalophilic floodwater mosquito involved in the transmission of several arboviruses. Rift Valley fever (RVF) is a relevant mosquito-borne zoonosis, affecting mainly humans and ruminants, that causes severe impact in public health and economic loses. Due to globalization and climate change, the European continent is threatened by its introduction. The main purpose of the present study was to evaluate the vector competence of a European field-collected Ae. vexans population. METHODS: Aedes vexans field-collected larvae were reared in the laboratory under field-simulated conditions. To assess the vector competence for Rift Valley fever phlebovirus (RVFV) transmission, adult F0 females were exposed to infectious blood meals containing the 56/74 RVFV strain. Additionally, intrathoracic inoculations with the same virus strain were performed to evaluate the relevance of the salivary gland barriers. Natural circulation of alphavirus, flavivirus and phlebovirus was also tested. RESULTS: To our knowledge, an autochthonous Ae. vexans population was experimentally confirmed as a competent vector for RVFV for the first time. This virus was capable of infecting and disseminating within the studied Ae. vexans mosquitoes. Moreover, infectious virus was isolated from the saliva of disseminated specimens, showing their capacity to transmit the virus. Additionally, a natural infection with a circulating Mosquito flavivirus was detected. The co-infection with the Mosquito flavivirus seemed to modulate RVFV infection susceptibility in field-collected Ae. vexans, but further studies are needed to confirm its potential interference in RVFV transmission. CONCLUSIONS: Our results show that field-collected European Ae. vexans would be able to transmit RVFV in case of introduction into the continent. This should be taken into consideration in the design of surveillance and control programmes.

European Aedes caspius mosquitoes are experimentally unable to transmit Zika virus.

BACKGROUND: Aedes caspius (Pallas, 1771) is a floodwater mosquito species widely distributed in the Western Palaearctic. As an anthropophilic species, its role as an arbovirus vector may be the key for understanding the transmission cycle of certain diseases in Europe such as Zika virus (ZIKV). Concerning vector competence for ZIKV, studies related to Ae. caspius are still scarce. ZIKV is an arbovirus that has provoked a widespread epidemic in the Pacific region (2007-2013) and in the Americas (2015-2016). ZIKV is associated with serious neurological injuries (e.g. microcephaly) and Guillain-Barré syndrome. Due to the ZIKV epidemics in the American continent, some viraemic travellers coming from endemic countries have been reported in Europe. More knowledge is therefore required to define the susceptibility of autochthonous mosquito species such as Ae. caspius for ZIKV in order to improve arbovirus surveillance and control programmes. In the present study, the vector competence of a European population of Ae. caspius was evaluated for two ZIKV lineages, the Suriname ZIKV strain (Asian lineage) and the MR766 ZIKV strain (African I lineage). Females were tested at 7, 14 and 21 days post-exposure (dpe) to infectious blood meals. An Ae. aegypti PAEA strain was used as a positive control. RESULTS: Aedes caspius presented low susceptibility to ZIKV infection and the virus was only detected by RT-qPCR in body samples. Low viral loads were detected for the MR766 strain at 7 dpe and for the Suriname strain at 14 and 21 dpe. Aedes caspius was unable to produce a disseminated infection and virus transmission at any of the tested time points. Using Ae. aegypti PAEA strain, infection, dissemination and transmission rates were calculated for the Suriname ZIKV strain (Asian lineage) at each time point. For the MR766 ZIKV strain (African I lineage), while only infection rates were estimated at each time point, no dissemination or transmission were detected in either species. CONCLUSIONS: The results of the present study reveal that the tested Ae. caspius population has a strong midgut escape barrier that limits the dissemination or transmission of the virus. As such, it seems unlikely that European Ae. caspius mosquitoes could be involved in ZIKV transmission if ZIKV was introduced into Europe. This information may help in designing a better strategy to European surveillance and control programmes for ZIKV.

Culex flavivirus infection in a Culex pipiens mosquito colony and its effects on vector competence for Rift Valley fever phlebovirus.

BACKGROUND: Rift Valley fever is a mosquito-borne zoonotic disease that affects domestic ruminants and humans. Culex flavivirus is an insect-specific flavivirus that naturally exists in field mosquito populations. The influence of Culex flavivirus on Rift Valley fever phlebovirus (RVFV) vector competence of Culex pipiens has not been investigated. METHODS: Culex flavivirus infection in a Cx. pipiens colony was studied by Culex flavivirus oral feeding and intrathoracical inoculation. Similarly, vector competence of Cx. pipiens infected with Culex flavivirus was evaluated for RVFV. Infection, dissemination, transmission rates and transmission efficiency of Culex flavivirus-infected and non-infected Cx. pipiens artificially fed with RVFV infected blood were assessed. RESULTS: Culex flavivirus was able to infect Cx. pipiens after intrathoracically inoculation in Cx. pipiens mosquitos but not after Culex flavivirus oral feeding. Culex flavivirus did not affect RVFV infection, dissemination and transmission in Cx. pipiens mosquitoes. RVFV could be detected from saliva of both the Culex flavivirus-positive and negative Cx. pipiens females without significant differences. Moreover, RVFV did not interfere with the Culex flavivirus infection in Cx. pipiens mosquitoes. CONCLUSIONS: Culex flavivirus infected and non-infected Cx. pipiens transmit RVFV. Culex flavivirus existing in field-collected Cx. pipiens populations does not affect their vector competence for RVFV. Culex flavivirus may not be an efficient tool for RVFV control in mosquitoes.

Genetic diversity of the HLA system in human populations from the Sierra (Andean), Oriente (Amazonian) and Costa (Coastal) regions of Ecuador.

We studied HLA class I (HLA-A, -B) and class II (HLA-DRB1, -DQB1) alleles by PCR-SSP based typing in a total of 1101 Ecuadorian individuals from three regions of the country, the Coastal region, the Andean region, and the Amazonian region, to obtain information regarding allelic and haplotypic frequencies and their linkage disequilibrium. We find that the most frequent HLA haplotypes with significant linkage disequilibrium in those populations are HLA-A*24∼B*35∼DRB1*04∼DQB1*03:02, A*02∼B*35∼DRB1*04∼DQB1*03:02, A*24∼B*35∼DRB1*14∼DQB1*03:01, A*02∼B*35∼DRB1*14∼DQB1*03:01 and A*02∼B*40:02∼DRB1*04∼DQB1*03:02. The only non-Native American haplotype with frequency >1% shared by all groups was A*29∼B*44∼DRB1*07∼DQB1*02. Admixture estimates obtained by a maximum likelihood method using HLA-B as genetic estimator revealed that the main genetic components for this sample of mixed-ancestry Ecuadorians are Native American (ranging from 52.86% to 63.83%) and European (from 28.95% to 46.54%), while an African genetic component was only apparent in the Coastal region (18.19%). Our findings provide a starting point for the study of population immunogenetics of Ecuadorian populations.